Rapid detection of bio-agents is required in a number of applications, including monitoring water supplies, protecting critical facilities, process control, and hazard analysis by first responders. These applications also require high sensitivity and high specificity (i.e., low false-alarm rate), and they place substantial demands on the physical characteristics (size, weight, power consumption), reliability, and cost of the instrument. Sensors based on microfluidic separation and analysis of aqueous samples have the potential to simultaneously address all of these needs.
Instruments based on microfluidic separation of complex mixtures have emerged as nearly ideal platforms for identification of a wide range of biological compounds. In particular, it is of current interest to rapidly detect pathogenic biological compounds which could be used as weapons. However, such species are typically present in very low concentration and if rapid detection is of interest, e.g., detection in less than 1 minute (such as for detect-to-warn scenarios), time-consuming sample processing must be eliminated. Unfortunately, most current methods used to achieve a low detection limit rely on ultra-sensitive detection methods such as laser-induced fluorescence (“LIF”) that require significant sample processing such as fluorescent tagging (e.g., U.S. Pat. No. 6,197,503, issued Mar. 6, 2001), to be effective. Other methods, therefore, are necessary.
In particular, the use of ring-down time of a light signal in a cavity (i.e., cavity ring-down spectroscopy or “CRDS”) can be used to measure optical characteristics of an absorbing medium. Such optical cavities consist of two or more mirrors between which an optical signal is reflected to characterize the mirrors as well as the optical characteristics of an absorbing medium (e.g., gases, molecular beams, etc.) between the mirrors (see for instance U.S. Pat. No. 5,528,040, issued Jun. 18, 1996). This technique has also been used for evanescent wave spectroscopy (U.S. Pat. No. 5,835,231, issued Nov. 10, 1998).
However, while a number of high-sensitivity absorption methods have been developed for gas-phase applications, condensed-phase analogs have been slow to emerge, Until recently, applications in condensed phase have been limited generally to absorption measurements of films through evanescent field experiments on the surface of all-solid state cavities and to films deposited on windows inside the cavity (see Pipino et al., Review of Scientific Instruments, 1997, v. 68, pp. 2978-2989; and Engeln et al., Journal of Chemical Physics, 1999, v. 110, pp. 2732-2733).
Beginning in 2002, however, the application of CRDS to absorption measurements on liquid samples began to be reported (see Hallock et al., Review of Scientific Instruments, 2002, v. 74, pp. 1741-1743; and Xu et al., Review of Scientific Instruments, 2002, v. 73, pp. 255-258) and more recently a CRDS device has been shown interfaced with a microfluidic separation column through which light was passed and collected using optical fibers (U.S. Pat. No. 6,842,548, issued Jan. 11, 2005).
Unfortunately, application of absorption-based detection in microfluidic systems has been limited by poor sensitivity due to the short path lengths (e.g., 10 μm to 100 μm) characteristic of the channel width of most microfluidic devices.
To overcome this problem, a sensing system is described which comprises a light transmitting/sensing means utilizing a cavity-ringdown laser for generating an evanescent field in a short length of optical fiber, and a microfluidic separation channel having a series of connected, prism segments for rotating and broadening the path of a flowing liquid while minimizing the dispersion of that flow such as is described in co-pending, commonly-owned U.S. patent application Ser. No. 10/456,772 now U.S. Pat. No. 7,005,301, herein incorporated by reference. The combination of these techniques can increase the effective absorption path length in the microfluidic channel by a factor of 103 or more, thereby providing the necessary sensitivity using direct absorption.